The first step in the activation of the prohormone, thyroxine (T4), is its 5' monodeiodination to T3, catalyzed by the types 1 and 2 iodothyronine deiodinase. Type 3 deiodinase (D3) catalyzes removal of an inner ring iodine inactivating T4 or T3. These three enzymes have two common features; they are integralmembrane proteins and all have the rare amino acid selenocysteine in their active center to serve as the iodine acceptor. They confer two general advantages to the regulation of thyroid hormone action. First, during embryological development, activation and inactivation of thyroid hormone is programmed to occur at specific times in specific cells to allow either induction or repression of thyroid hormone-dependent gene expression. Second, throughout life, the important role of D2 in the hypothalamic-pituitary-thyroid axis permits the terrestrial vertebrate to adapt to iodine deficiency by monitoring the concentration of T4 in the circulation. This permits increases in TRH and TSH secretion, long before the concentrations of the active hormone, T3, decrease. In humans, it has long been assumed that D1, present in high concentrations in the liver and kidney, is the major source of circulating T3. More recent analyses of D2 expression patterns together with older in vivo results suggest that this is not the case and that most plasma T3 in humans probably derives from the activation of T4 by D2. This enzyme is widely expressed in humans, but not in rodents. The present proposal will focus on 3 aspects of the molecular physiology of the deiodinases. Specific Aim I will analyze the causes of the unique topology and subcellular localizations of D1, D2 andD3 in the cell using confocal microscopy and cell biological techniques. Specific Aim II will employ cells expressing endogenous or recombinant D1 and D2 to understand why D2 is the major enzyme catalyzing extrathyroidal T3 production in humans. We also will determine why the T3 produced by D2-catalyzed T4 5'- deiodination enters the nucleus whereas that generated by D1 does not. Specific Aim III will address the issue of the active form of the three enzymes. Are they homodimers or do they interact with other proteins? We will also analyze the functional effects of this interaction. These studies will thus address the basic mechanisms by which thyroid hormone activation and inactivation are regulated in both normal and pathological states.